The present invention is related to the construction and application of recombinant vaccinia virus containing DNA sequences for encoding and efficient expression of enzymatically active cytochromes P-450 in mammalian cells.
The cytochromes P-450 are a large family of hemoproteins capable of metabolizing xenobiotics such as drugs, carcinogens and environmental pollutants as well as endobiotics such as steroids, fatty acids and prostaglandins. Some members of the cytochrome P-450 family are inducible in both animals and cultured cells while other constitutive forms are non-inducible. This group of enzymes carry out beneficial metabolic activities by detoxification of xenobiotics as well as harmful metabolic conversion of xenobiotics to toxic, mutagenic and carcinogenic forms (Gelboin, Physiol.Rev. 60:1107-1166, 1980).
In animals, multiple molecular forms of cytochrome P-450s are expressed simultaneously and they all exhibit common physical and biological properties. The multiplicity and common properties of the cytochromes P-450 make it difficult to separate different forms of cytochrome P-450 especially the minor forms. And even where P-450 cytochromes may have been obtained perhaps in purified form by conventional enzyme purification procedures, they were obtained out of the natural biological membrane association and all such preparations require the addition of NADPH-cytochrome P-450 reductase and other cell fractions for enzymatic activity. These factors have prevented a clearer understanding of the role and function of individual forms of cytochrome P-450 in metabolism, detoxification and activation of both xenobiotic and endobiotic substrates. Recent reports on the expression of cloned P-450 DNA sequences by DNA transfection are limited to yeast and COS 1 monkey helper cells (Oeda et al, and Zuber et al, DNA, 4:203,210, 1985; Science 234:1258-1261, 1986). However, no expression of functional cytochrome P-450 has been reported in mammalian systems of wide host range using infectious viruses.